Ability of Bicarbonate Supplementation to Sensitize Selected Methicillin-Resistant Staphylococcus aureus (MRSA) Strains to β-Lactam Antibiotics in an Ex Vivo Simulated Endocardial Vegetation Model

Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth [CAMHB]) with bicarbonate (NaHCO3) significantly increases β-lactam susceptibility of selected MRSA strains (“NaHCO3-responsive”). This “sensitization” phenomenon translated to enhanced β-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO3-mediated β-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. Four previously described MRSA strains were used: two each exhibiting in vitro “NaHCO3-responsive” or “NaHCO3-nonresponsive” phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were evaluated in CAMHB±NaHCO3. Intra-SEV MRSA killing was determined over 72 hr exposure. In both NaHCO3-responsive strains, supplementation with 25 mM or 44 mM NaHCO3 significantly reduced β-lactam MICs to below the OXA susceptibility breakpoint (≤ 4 mg/L) resulting in bactericidal activity (≥ 3 log kill) in the model for both OXA and CFZ. In contrast, neither in vitro-defined NaHCO3-nonresponsive MRSA strains showed significant sensitization in the SEV model to either β-lactam. At both NaHCO3 concentrations, the fractional time-above-MIC was >50% for both CFZ and OXA in the NaHCO3-responsive MRSA. Also, in RPMI+10% LB media (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO3), both CFZ and OXA exhibited enhanced bactericidal activity against each NaHCO3-responsive strain in the SEV model. Neither CFZ nor OXA exposures selected for high-level β-lactam-resistant mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO3 supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate similar enhanced susceptibility in NaHCO3-supplemented media in vitro.


Supplementation of standard growth media (cation-adjusted Mueller-Hinton Broth 27
[CAMHB]) with bicarbonate (NaHCO 3 ) significantly increases β-lactam susceptibility of selected 28 MRSA strains ("NaHCO 3 -responsive"). This "sensitization" phenomenon translated to enhanced 29 β-lactam efficacy in a rabbit model of endocarditis. The present study evaluated NaHCO 3 -30 mediated β-lactam MRSA sensitization using an ex vivo pharmacodynamic model, featuring 31 simulated endocardial vegetations (SEVs), to more closely mimic the host microenvironment. 32 Four previously described MRSA strains were used: two each exhibiting in vitro "NaHCO 3 -33 responsive" or "NaHCO 3 -nonresponsive" phenotypes. Cefazolin (CFZ) and oxacillin (OXA) were 34 evaluated in CAMHB±NaHCO 3 . Intra-SEV MRSA killing was determined over 72 hr exposure. In 35 both NaHCO 3 -responsive strains, supplementation with 25 mM or 44 mM NaHCO 3 significantly 36 reduced β-lactam MICs to below the OXA susceptibility breakpoint (≤ 4 mg/L) resulting in 37 bactericidal activity (≥ 3 log kill) in the model for both OXA and CFZ. In contrast, neither in vitro-38 defined NaHCO 3 -nonresponsive MRSA strains showed significant sensitization in the SEV 39 model to either β-lactam. At both NaHCO 3 concentrations, the fractional time-above-MIC was 40 >50% for both CFZ and OXA in the NaHCO 3 -responsive MRSA. Also, in RPMI+10% LB media 41 (proposed as a more host-mimicking microenvironment and containing 25 mM NaHCO 3 ), both 42 CFZ and OXA exhibited enhanced bactericidal activity against each NaHCO 3 -responsive strain 43 in the SEV model. Neither CFZ nor OXA exposures selected for high-level β-lactam-resistant 44 mutants within SEVs. Thus, in this ex vivo model of endocarditis, in the presence of NaHCO 3 45 supplementation, both CFZ and OXA are highly active against MRSA strains that demonstrate 46 similar enhanced susceptibility in NaHCO 3 -supplemented media in vitro. 47

INTRODUCTION
resulting minimum inhibitory concentrations (MICs) may not accurately represent antibiotic 74 activity against MRSA within host-specific microenvironments (13). 75 Recent studies have focused on refining in vitro growth media in order to better simulate 77 the host microenvironment in vivo in the context of more relevant and translatable antimicrobial 78 susceptibility testing. Supplementation of standard media with bicarbonate (NaHCO 3 ), a 79 ubiquitous buffer in humans, has become the subject of several such studies (13,14). These 80 reports have demonstrated the ability of NaHCO 3 to alter the susceptibility of selected MRSA 81 strains in vitro to β-lactams. These investigations have focused on two conventional, prototype 82 β-lactams not recommended for use against MRSA, OXA and cefazolin (CFZ) (14). These data 83 enabled identification of two distinct MRSA phenotypes, NaHCO 3 -"responsive" and NaHCO 3 -84 "nonresponsive". These in vitro phenotypes accurately predicted the ability of these same 85 β-lactams to clear MRSA from multiple target tissues in a rabbit model of MRSA infective 86 endocarditis (i.e., cardiac vegetations, kidneys and spleen) (14). The mechanism(s) by which 87 NaHCO 3 sensitizes "responsive" strains to such β-lactams appears to involve, at least in part, 88 multiple genes which are critical in maintaining the MRSA phenotype, such as mecA and sarA 89 (12); these perturbations may, in turn, lead to decreased production and/or maturation of 90 PBP2a, yielding a "functional MSSA" phenotype (14). 91

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The present study expands on our previous findings of bicarbonate sensitization of 93 MRSA to β-lactams using a pharmacodynamic model featuring ex vivo simulated endocardial 94 vegetations (SEVs). We hypothesized that 'bicarbonate responsiveness' in MRSA in this model 95 would mirror similar findings in the rabbit endocarditis model, and that host-mimicking media 96 within SEVs would help identify novel pharmacodynamic optimization strategies for prototypical 97 β-lactams against such NaHCO 3 -responsive MRSA strains. 98 RESULTS 100 β-lactam susceptibilities in standard and alternative media supplemented with 101 NaHCO 3 . CFZ and OXA MICs have been previously reported for these four study strains (14). 102 In this investigation, we confirmed the strain-dependent NaHCO 3 enhancement of the 103 susceptibility of these isolates to the two study β-lactams. As noted in Table 1, strains 11-11 104 (USA300) and MW2 (USA400) displayed a significant reduction in β-lactam MICs in media 105 supplemented with NaHCO 3 (44 mM), while β-lactam susceptibility was not affected with 106 NaHCO 3 supplementation for the other two strains, COL (USA100) and BMC-1001 (USA500). 107 In the two NaHCO 3 -responsive strains, supplementation with 44 mM NaHCO 3 reduced the CFZ 108 MIC to below the OXA susceptibility breakpoint (≤ 4 mg/L), with an 8-16-fold MIC reduction. 109 Similarly, for these same strains, the OXA MICs were reduced 16-64-fold with 44 mM NaHCO 3 110 supplementation. 111 112 It has recently been reported that an 'antibiotic sensitization' effect, especially for gram-113 negative bacteria and selected β-lactams, can occur in other host-mimicking media (13-15). The 114 standard cell culture media, RPMI 1640, contains physiologic concentrations of NaHCO 3 115 (~25 mM) . In this media, as opposed to CA-MHB, we noted substantially increased β-lactam 116 susceptibility in all four strains regardless of genotypic background. However, the two NaHCO 3 -117 responsive strains were generally more responsive in this host-mimicking media, with resultant 118 β-lactam MICs of ≤ 2 mg/L (Table 1), consistent with previous findings (14). 119 120 β-lactam pharmacokinetics in the ex vivo SEV model. The pharmacokinetic profiles 121 of antibiotics within the ex vivo SEV model is computer designed to precisely mimic actual 122 patient exposures clinically.  Overall, there was a notable NaHCO 3 -concentration response, with higher bacterial 165 count reductions, faster time to bactericidal activity (time to 3 log 10 CFU/g reduction), and lower 166 AUBC when media was supplemented with 44 mM vs 25 mM NaHCO 3 (Figure 1; Table 3. This 167 correlated with greater susceptibility with the higher NaHCO 3 concentration, resulting in 100% 168 ƒT>MIC for both the 11-11 and MW2 strains with 44 mM NaHCO 3 vs 57% and 83% ƒT>MIC, 169 respectively, with 25 mM NaHCO 3 for these strains. Since no change in MIC occurred in 170 CAMHB + NaHCO 3 in the nonresponsive strains, the ƒT>MIC remained at zero percent, and 171 reflects the lack of any β-lactam activity against those strains. 172 173  CFZ and OXA are each bactericidal ex vivo against NaHCO 3 -responsive (but not 174 against non-responsive) MRSA in the host-mimicking medium, RPMI. As noted above, CFZ and 175 OXA MICs for all four strains were substantially reduced in RPMI. We next determined if these 176 ,in vitro outcomes were mirrored ex vivo in the SEV model. As displayed in Figure 3, the 177 NaHCO 3 -responsive strains, 11-11 and MW2, were significantly killed with CFZ or OXA 178 treatment in this medium; this is reflected in the enhanced pharmacodynamic attainment in this 179 medium, resulting in 100% ƒT>MIC for CFZ and 60% for OXA against both 11-11 and MW2 180 strains. In contrast, over the same 72 hrs β-lactam exposure period, the two nonresponsive 181 strains (COL and BMC-1001) were minimally affected by either agent. It should be noted that 182 there was initial activity with CFZ against COL in RPMI, with ~2 log 10 CFU/g killing at 24-48 hrs. 183 However, this was not sustained after 48 hours, and the strain regrew to the initial inoculum. 184 These data are reflected in the lower target attainment of 8.2%-56% ƒT>MIC for CFZ and 0%-185 20% for ƒT>MIC for OXA. 186 187  β-lactam treatment did not select for high-level CFZ or OXA resistance 188 regardless of the NaHCO 3 -responsivity phenotype. One additional advantage of this ex vivo 189 model system is the ability to screen for emergence of drug-resistant mutants during human- The β-lactam class of antibiotics remains the treatment of choice for a broad range of 206 susceptible pathogens due to their potent and rapid mechanism(s) of action, as well as their 207 relatively low rates of adverse side effects and toxicities. In addition, they have recently become 208 well-characterized as able to augment the innate host immune system, via their synergistic 209 interactions with host defense peptides against key gram-positive pathogens, including S. 210 aureus (19)(20)(21). 211

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The current study extends our previous work on the ability of NaHCO 3 supplementation 213 of standard MRSA in vitro media to identify a sub-set of MRSA strains that may, in fact, exhibit 214 β-lactam susceptibility (14,22,23). The notion of employing alternative media for antibiotic 215 susceptibility screening is not new; however, the biological basis and clinical utility of this 216 approach has substantially increased in the last few years. For example, Kubicek-Sutherland et 217 al mimicked the intracellular conditions of the macrophage phagosome in which Salmonella 218 resides, using a mildly acidic and phosphate/magnesium-poor culture medium. Their study 219 revealed that when Salmonella were grown in this media, antimicrobial susceptibility profiles 220 emerging from this scenario were better predictors of treatment outcomes in murine bacteremia 221 models than more traditional susceptibility testing strategies (23). Similar studies using 222 Acinetobacter and staphylococci grown in Dulbecco Modified Eagles Medium (DMEM) or RPMI 223 were better predictors of in vivo treatment outcomes, when compared to standard susceptibility 224 testing modalities (13, 15). It should be emphasized that all four of our prototype MRSA isolates 225 were rendered significantly more β-lactam-susceptible in vitro in RPMI media (4-to 64-fold MIC 226 reductions). However, these salutary in vitro results in RPMI did not accurately predict 227 subsequent microbiologic outcomes in the SEV model. 228

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The enhanced antimicrobial activity seen above in such alternative media, as well as 230 their outcome predictability in vivo may be, in part, reflective of: i) the enhanced activity of host 231 defense peptides in combination with antibiotics in vivo; ii) bacterial adaptations in vivo that 232 prevent excessive microbial growth; and/or iii) blunted expression of antimicrobial resistance 233 mechanisms (14,15,20). However, it should be noted that some host-mimicking environments 234 may actually be beneficial for bacterial survival. For example, under more acidic conditions, E. 235 coli may become more resistant to β-lactams due to higher PBP1b production needed for 236 organism survival in this harsh environment (24). 237

238
The treatment of MRSA infections poses several daunting challenges. Current 239 treatment options for MRSA infections are relatively limited, and include antibiotics that are not 240 only costly, but also substantially less effective, and often more toxic when compared to 241 standard antibiotic treatments for MSSA (25)(26)(27). This is in large part due to the perceived 242 inability to use traditional β-lactam antibiotics for MRSA infections (6). The mecA gene is 243 primarily responsible for mediating S. aureus resistance to traditional β-lactams, encoding for 244 penicillin-binding protein 2A (PBP2A), which has low affinity to most standard-of-care 245 antistaphylococcal β-lactam antibiotics (6). Despite this, combination therapy featuring β-246 lactams plus anti-MRSA agents such as vancomycin and daptomycin have proven to be 247 effective in selected patients with recalcitrant MRSA infections (19,(28)(29)(30). In addition, many 248 MRSA strains which exhibit reduced susceptibility to vancomycin and daptomycin often 249 demonstrate a paradoxical increase in susceptibility to β-lactams, a phenomenon known as the 250 "see-saw effect"; this provides an additional scenario in which β-lactam agents may provide 251 synergistic efficacy for MRSA killing (19). 252 253 Following our discovery of the NaHCO 3 responsiveness of several prototype MRSA 254 strains in terms of β-lactam hypersusceptibility in vitro (14), we recently screened a large 255 collection of well-characterized MRSA strains (n = 58) for this same phenotype. We identified 256 that ~three-quarters of this cohort were CFZ-susceptible in NaHCO 3 -supplemented CA-MHB, 257 while ~one-third were OXA-susceptible in the same media (31). We have investigated the 258 potential mechanisms by which NaHCO 3 may cause such β-lactam hypersusceptibility in vitro 259 and demonstrated that NaHCO 3 -supplemented media can down-regulate expression of the 260 mecA gene, and subsequent PBP2a production, as well as by impacting several genes involved 261 in maintenance of the MRSA phenotype, including sarA and blaZ (14). 262

263
The pharmacodynamic parameter best correlated with efficacy of β-lactam antibiotics is 264 ƒT>MIC (time-above-MIC of the free drug); this metric has been highly studied in vivo and in 265 vitro, and is validated to predict improved clinical outcomes in β-lactam-treated patients (32)(33)(34)(35). 266 In most investigations, β-lactams demonstrate optimal activity with ƒT>MIC in the range of 40-267 60% (32). Relevant to our study, we found that the lower MICs observed in NaHCO 3 -268 supplemented media for two of our prototype strains (11-11 and MW2) resulted in an "MSSA-269 like phenotype" improving the potential for target attainment for both CFZ and OXA. For the 270 NaHCO 3 -responsive strains, CFZ achieved at least 83% ƒT>MIC, resulting in a substantial and 271 durable bactericidal activity in the ex vivo SEV model. Similarly, OXA, with a calculated ~60% 272 ƒT>MIC in the two NaHCO 3 -responsive strains, also achieved good bactericidal activity in the 273 ex vivo model. 274

275
The ex vivo simulated endocardial vegetation (SEV) model has been extensively used to 276 pharmacokinetically and pharmacodynamically study the impacts of many antimicrobials against 277 several clinical bacterial isolates in order to verify these interactions in a setting more akin to the 278 host tissue microenvironment than in vitro assays (36, 37). In addition, this model has been 279 shown to successfully recapitulate the microbiologic results generated in several in vivo animal 280 models, including rabbit endocarditis (IE) (38). In the present investigation, we used this same 281 ex vivo pharmacokinetic-pharmacodynamic model to more systematically evaluate the activity of 282 CFZ and OXA against our four prototype MRSA isolates, in a host-mimicking microenvironment. 283 Our current results in the SEV model provided an important "bridge" between in vitro and in vivo 284 outcomes with these strains. First, the two MRSA that were significantly β-lactam/NaHCO 3 -285 responsive in vitro and in vivo (14), showed enhanced intra-SEV killing by CFZ and OXA in the 286 presence of NaHCO 3 . In parallel, the two β-lactam/NaHCO 3 -nonresponsive strains (as defined 287 in vitro and in vivo) were not substantially killed by CFZ or OXA ex vivo. Second, as seen in 288 vitro, there appeared to be a NaHCO 3 concentration-optimum for the β-lactam/NaHCO 3 289 sensitizing phenotype for the two responsive strains (11-11 and MW2); thus, significant killing There remains a current debate over the most appropriate β-lactam to treat serious 298 MSSA infections, with CFZ or OXA as the primary candidates (25, 39); most recent MSSA 299 bacteremia studies appear to favor CFZ (16,26,27). Further, on balancing adverse events vs 300 efficacy metrics, CFZ is generally regarded as a safer option, due to its decreased renal toxicity 301 risk as compared to the antistaphylococcal penicillins (16, 27). However, there remains concern 302 about CFZ efficacy in "high-inoculum" S. aureus infections (e.g., endocarditis) due to the 303 potential for the undetected presence and induction of genes for Types A or C 304 cephalosporinases that hydrolyze CFZ (39,40). In our current study, CFZ appeared to be 305 unaffected by the high inoculum within SEVs, at least for the NaHCO 3 -responsive strains, 11-11 306 and MW2, with excellent CFZ bactericidal activity obtained despite starting inocula ≥ 1x10 8 307 CFU/g. 308 309 310 In conclusion, this study further validates the potential clinical translatability of the 311 intriguing finding of β-lactam/NaHCO 3 sensitization of selected MRSA strains in vitro. It will be 312 important to extend our studies to even larger clinical MRSA cohorts (31). Ultimately, a pivotal 313 clinical trial will be required to fully adjudicate the clinical utility of defining MRSA strains as 314 β-lactam/NaHCO 3 -responsive in the clinical microbiology laboratory. weighing ~0.7 g and containing 3-3.5 g/dL of albumin and 6.8-7.4 g/dL of total protein (equating 361 to human physiologic levels). The protein binding of the study drugs has been found to be 84% 362 for CFZ and 92% for OXA, which was used to calculate free AUC (ƒAUC) and percent time of 363 free drug above the MIC (%ƒT>MIC) (41,42).